Preclinical safety and efficacy of andexanet alfa in animal models.

Essentials There is currently no approved reversal agent for factor Xa (FXa) inhibitors Andexanet alfa has been developed to reverse the anticoagulant effects of FXa inhibitors Andexanet reduced blood loss and anticoagulation markers in rivaroxaban-anticoagulated rabbits Andexanet was well tolerated in monkeys and rats, with no evidence of prothrombotic activity SUMMARY: Background Andexanet alfa is a recombinant modified form of factor Xa (FXa), designed to bind to and reverse the anticoagulant activity of FXa inhibitors. Objectives To evaluate the ability of andexanet to reverse the anticoagulant activity of rivaroxaban, and assess its pharmacokinetics (PK) and toxicity in animal models. Methods The effects of andexanet on blood loss, anti-FXa activity, rivaroxaban unbound plasma concentrations and other coagulation parameters were assessed in a rabbit liver laceration 'treatment' model. Andexanet was administered 10 min after blood loss was initiated. The toxicity of repeated administration of andexanet (up to 60 mg kg-1 day-1 ) was assessed in cynomolgus monkeys. PK parameters were evaluated in rats and monkeys. Results Excess blood loss due to anticoagulation with rivaroxaban was significantly decreased by a single intravenous bolus administration of andexanet at 35 and 75 mg per rabbit, by 75% and 63%, respectively. This correlated with dose-dependent decreases in the unbound fraction of rivaroxaban and anti-FXa activity. Co-administration of rivaroxaban had no significant impact on the PK parameters of andexanet. Andexanet (up to 60 mg kg-1 day-1 ) was well tolerated in monkeys, with no accumulation of andexanet or rivaroxaban. There was a single occurrence of anaphylaxis, which resolved after treatment with diphenhydramine and epinephrine. There was no histological evidence of prothrombotic activity with high-dose andexanet compared with vehicle control, as measured by clot and fibrin deposition in all major organs. Conclusions These data suggest that andexanet is a promising therapy for the reversal of FXa inhibitor-induced anticoagulation, supporting clinical studies in humans.

Age-related and tissue-specific accumulation of oxidative DNA base damage in 7,8-dihydro-8-oxoguanine-DNA glycosylase (Ogg1) deficient mice.

Mutations that influence the repair of oxidative DNA modifications are expected to increase the steady-state (background) levels of these modifications and thus create a mutator phenotype that predisposes to malignant transformation. We have analysed the steady-state levels and repair kinetics of oxidative DNA modifications in cells of homozygous ogg1(-/-) null mice, which are deficient in Ogg1 protein, a DNA repair glycosylase that removes the miscoding base 8-hydroxyguanine (8-oxoG) from the genome. Oxidative purine modifications including 8-oxoG were quantified by means of an alkaline elution assay in combination with Fpg protein, the bacterial functional analogue of Ogg1 protein. In primary hepatocytes of adult ogg1(-/-) mice aged 9-12 months, the steady-state level of the lesions was 2.8-fold higher than in wild-type control mice. In contrast, no difference between ogg1(-/-) and wild-type mice was observed in splenocytes, spermatocytes and kidney cells. In hepatocytes of ogg1(-/-) mice, but not in wild-type controls, the steady-state levels increased continuously over the whole lifespan. No significant accumulation of the oxidative base modifications was observed in ogg1(-/-) fibroblasts in culture when they were kept confluent for 8 days. Both in confluent and proliferating ogg1(-/-) fibroblasts, the global repair of additional oxidative base modifications induced by photosensitization was 4-fold slower than in wild-type cells. The results suggest that the consequences of an Ogg1 defect are restricted to slowly proliferating tissues with high oxygen metabolism such as liver, because of a back-up mechanism for the repair of 8-oxoG residues that is independent of transcription and replication.

Efficacy of the novel selective platelet-derived growth factor receptor antagonist CT52923 on cellular proliferation, migration, and suppression of neointima following vascular injury.

Exaggerated or inappropriate signaling by the platelet-derived growth factor receptor (PDGFR) tyrosine kinase has been implicated in a wide variety of diseases. Thus, a series of piperazinyl quinazoline compounds were identified as potent antagonists of the PDGFR by screening chemical libraries. An optimized analog, CT52923, was shown to be an ATP-competitive inhibitor that exhibited remarkable specificity when tested against other kinases, including all members of the closely related PDGFR family. The PDGFRs and stem cell factor receptor were inhibited with an IC(50) of 100 to 200 nM, while 45- to >200-fold higher concentrations of CT52923 were required to inhibit fms-like tyrosine kinase-3 and colony-stimulating factor-1 receptor, respectively. Other receptor tyrosine kinases, cytoplasmic tyrosine kinases, serine/threonine kinases, or members of the mitogen-activated protein kinase pathway were not significantly inhibited at 100- to 1000-fold higher concentrations. In addition, this compound also demonstrated specificity for inhibition of cellular responses. Platelet-derived growth factor-induced smooth muscle cell migration or fibroblast proliferation was found to be blocked by CT52923 with an IC(50) of 64 and 280 nM, respectively, whereas 50- to 100-fold higher concentrations were required to inhibit these responses when induced with fibroblast growth factor. To investigate the effect of CT52923 on PDGFR signaling, in vivo studies demonstrated that CT52923 could significantly inhibit neointima formation following carotid artery injury by oral administration in the rat. Therefore, PDGFR antagonism by CT52923 could be a viable strategy for the prevention of clinical restenosis or the treatment of other human diseases involving PDGFR signaling.

Design and synthesis of glycolic and mandelic acid derivatives as factor Xa inhibitors.

A series of glycolic and mandelic acid derivatives was synthesized and investigated for their factor Xa inhibitory activity. These analogues are highly potent and selective inhibitors against fXa. In a rabbit deep vein thrombosis model, compound 26 showed significant antithrombotic effects (81% inhibition of thrombus formation) at 1.1 microM plasma concentration following intravenous administration.

Discovery of transition state factor Xa inhibitors as potential anticoagulant agents.

Factor Xa is an attractive biological target in the discovery and development of either parenteral or orally active anticoagulant agents. Several strategies have been utilized at COR Therapeutics in the pursuit of tri-peptide based transition state mimetic factor Xa inhibitors with high aqueous solubility. Some of these inhibitors have displayed excellent in vitro potency in inhibiting factor Xa in the prothrombinase complex. More importantly, these compounds showed strong in vivo antithrombotic efficacy without significant bleeding complications in several animal thrombosis models. These results demonstrated that small molecule factor Xa inhibitors could be advantageous over Warfarin and LMWH. For the discovery and development of orally active anticoagulant agents, small organic molecules as reversible factor Xa inhibitors were explored. From a medicinal chemistry perspective, significant insight has been gained regarding the in vivo antithrombotic efficacy and pharmacokinetic behaviors of each class of factor Xa inhibitors. This review will focus on the design and discovery of transition state factor Xa inhibitors as potential parenteral anticoagulant agents. Several excellent comprehensive review articles on factor Xa inhibitors have appeared recently [1-4].

Antithrombotic and hemostatic capacity of factor Xa versus thrombin inhibitors in models of venous and arteriovenous thrombosis.

Thrombin plays a central role in venous and arterial thrombosis. We utilized two different rabbit models of in vivo thrombosis to investigate the effect of inhibitors of thrombin generation and thrombin activity. The agents tested were specific inhibitors of factor Xa (fXa) [N2-[(phenylmethyl)sulfonyl]-D-arginyl-N-[(1S)-4-[(aminoiminomethyl++ +)a mino]-1-(2-thiazolylcarbonyl)butyl]-glycinamide (C921-78)] and thrombin [D-phenylalanyl-N-[4-[(aminoiminomethyl)amino]-1-(chloroacetyl)but yl]-L-prolinamide (PPACK)], as well as drugs that affect both thrombin and fXa, unfractionated and low molecular weight (enoxaparin) heparin. The agents administered as constant intravenous infusion were evaluated for antithrombotic efficacy in anesthetized rabbits. All four agents were capable of dose dependent inhibition of thrombosis in venous and arteriovenous thrombosis models. However, due to the more aggressive nature of thrombotic stimulation in the arteriovenous shunt model, complete cessation of thrombus growth was not achieved for any of the agents at the doses tested. Comparison between the agents focused on the differences in extension of coagulation parameters (activated partial thromboplastin time, prothrombin time, thrombin clotting time), changes in hematological parameters, and extension of rabbit cuticle bleeding time at doses required to produce maximum inhibition in the thrombosis models. In the venous thrombosis model at the maximally effective dose, C921-78 had minimal extension of ex vivo clotting parameters, while enoxaparin and unfractionated heparin demonstrated a two to sevenfold increase in activated partial thromboplastin times, and PPACK had a threefold extension of thrombin clotting times. In addition, unlike the other three agents, which exhibited no significant changes in hematological parameters, PPACK demonstrated dose dependent thrombocytopenia. A standardized cuticle bleeding time was used as a measure of perturbation of hemostasis. The agents were evaluated for significant increases in bleeding time at doses up to eight times that needed to completely inhibit venous thrombus formation. Unfractionated heparin displayed a significant bleeding time effect at the dose required to inhibit venous thrombosis (100 u/kg+2 u/kg/min). Enoxaparin and PPACK caused significant bleeding time extensions at four times the fully efficacious venous dose (800 u/kg+8 u/kg/min and 30 microg/kg/min). By contrast, C921-78 did not significantly increase bleeding time even at eight times the maximally effective dose (240 microg/kg+7.2 microg/kg/min). Our results demonstrate that specific inhibition of fXa can be utilized to derive potent antithrombotic activity without disrupting extravascular hemostasis.

Accumulation of premutagenic DNA lesions in mice defective in removal of oxidative base damage.

DNA damage generated by oxidant byproducts of cellular metabolism has been proposed as a key factor in cancer and aging. Oxygen free radicals cause predominantly base damage in DNA, and the most frequent mutagenic base lesion is 7,8-dihydro-8-oxoguanine (8-oxoG). This altered base can pair with A as well as C residues, leading to a greatly increased frequency of spontaneous G.C-->T.A transversion mutations in repair-deficient bacterial and yeast cells. Eukaryotic cells use a specific DNA glycosylase, the product of the OGG1 gene, to excise 8-oxoG from DNA. To assess the role of the mammalian enzyme in repair of DNA damage and prevention of carcinogenesis, we have generated homozygous ogg1(-/-) null mice. These animals are viable but accumulate abnormal levels of 8-oxoG in their genomes. Despite this increase in potentially miscoding DNA lesions, OGG1-deficient mice exhibit only a moderately, but significantly, elevated spontaneous mutation rate in nonproliferative tissues, do not develop malignancies, and show no marked pathological changes. Extracts of ogg1 null mouse tissues cannot excise the damaged base, but there is significant slow removal in vivo from proliferating cells. These findings suggest that in the absence of the DNA glycosylase, and in apparent contrast to bacterial and yeast cells, an alternative repair pathway functions to minimize the effects of an increased load of 8-oxoG in the genome and maintain a low endogenous mutation frequency.

Overexpression of Ogg1 in mammalian cells: effects on induced and spontaneous oxidative DNA damage and mutagenesis.

Chinese hamster ovary cell lines (AA8 and AS52) were stably transfected to overexpress hOgg1 protein, the human DNA repair glycosylase for 7,8-dihydro-8-oxoguanine (8-oxoG). In the transfectants, the repair rate of 8-oxoG residues induced by either potassium bromate or the photosensitizer [R]-1-[(10-chloro-4-oxo-3-phenyl-4H-benzo[a]quinolizin-1-yl)-carbo nyl ]-2-pyrrolidinemethanolplus light was up to 3-fold more rapid than in the parental cells. However, the improved repair had little effect on the mutagenicity of potassium bromate in the guanine phosphoribosyl transferase (gpt) locus of the OGG1-transfected AS52 cells. The steady-state (background) levels of DNA base modifications sensitive to Fpg protein, which include 8-oxoG, in cells not exposed to a damaging agent were not reduced by the overexpression of Ogg1 protein. Moreover, the spontaneous mutation rates in the gpt locus were similar in OGG1-transformed and vector-only-transformed cells. The results demonstrate the potential of Ogg1 protein to remove its substrate modifications from most of the chromosomal DNA. They indicate, on the other hand, that the Ogg1 protein alone may not be rate limiting for the repair of the residual substrate modifications observed in cells under normal growth conditions.

Integrilin prevents prolonged bleeding times after cardiopulmonary bypass.

BACKGROUND: Cardiopulmonary bypass reduces platelet number and function, increases postoperative bleeding time, and is the major, unsolved cause of nonsurgical bleeding after open heart operations. Temporary inhibition of platelet function during cardiopulmonary bypass (platelet anesthesia) protects platelets and reduces postoperative bleeding time and bleeding. METHODS: Integrilin, a short-acting, reversible platelet glycoprotein IIb/IIIa inhibitor was studied in 28 baboons that had 60 minutes of normothermic cardiopulmonary bypass using peripheral cannulas. A control group, two groups that received different doses of Integrilin, and a group that received a combination of Integrilin and low-dose Iloprost were studied. Blood samples for platelet count, aggregation to adenosine diphosphate, beta-thromboglobulin, prothrombin fragment F1.2, thrombin-antithrombin complex, and fibrinopeptide A were obtained at seven time points. Template bleeding times were measured before and at five intervals after cardiopulmonary bypass. RESULTS: Both doses of Integrilin and the combination of Integrilin and Iloprost significantly protected platelet number, inhibited the response to adenosine diphosphate, and reduced postoperative bleeding times, but they did not reduce beta-thromboglobulin release except in the high-dose Integrilin group. Thrombin formation and activity were qualitatively, but not significantly, reduced in all treatment groups. Bleeding times were not significantly different from baseline at the time protamine was given in the combination group and 60 minutes after protamine administration in all treatment groups. CONCLUSIONS: Integrilin alone or in combination with Iloprost significantly reduces platelet activation during cardiopulmonary bypass and produces normal or near-normal bleeding times at the time protamine is given.

Relative efficacy of active site-blocked factors IXa, Xa in models of rabbit venous and arterio-venous thrombosis.

In order to investigate the respective roles of prothrombinase and intrinsic tenase (IXa/VIIIa) in venous thrombosis, we compared the anti-thrombotic efficacy of inhibitors of these two coagulation complexes. The agents tested were dansyl-Glu-Gly-Arg chloromethyl ketone-inactivated bovine factor IXa(IXai) and Glu-Gly-Arg chloromethyl ketone-inactivated human factor Xa (Xai). In vitro formation of active complexes (prothrombinase or tenase) was inhibited by Xai and IXai resulting in IC50 values of 3 nM and 5 nM, respectively. Antithrombotic activity was measured by inhibition of clot accretion on cotton threads placed in the abdominal vena cava of anesthetized rabbits. Intravenous bolus dosing followed by infusion of Xai during the experimental protocol resulted in a dose dependent reduction of clot weight, a dosage of 16.0 micrograms/kg + 0.28 microgram/kg/min being sufficient to produce a 96% inhibition of thrombosis. A much higher dose of IXai (1.0 mg/kg + 17.3 micrograms/kg/min) resulted in a 39% reduction of clot weight. In a rabbit arterio-venous shunt model mimicking arterial thrombosis, the relative efficacy of the two agents was found to be more comparable. The doses required for optimum antithrombotic activity were 128.0 micrograms/kg + 2.2 micrograms/kg/min for Xai and 1.0 mg/kg + 17.3 micrograms/kg/min for IXai. We conclude that, in this study, prothrombinase rather than tenase inhibition was more effective in reducing venous thrombosis and that these effects can be achieved without disruption of extravascular hemostasis.

Procoagulant activity of reversibly acylated human factor Xa.

The plasma clotting factors used to treat hemophiliacs who have developed inhibitory antibodies have a shared history of limited clinical safety and utility. To improve on existing bypass factors, we have developed a reversibly acylated form of human plasma factor Xa capable of providing a time-dependent release of procoagulant activity. Factor Xa was treated with p-amidinophenyl p'-anisate to generate anisoyl Xa. The chemical modification of the protein involves acylation of the active site serine residue of factor Xa. Anisoyl Xa deacylated in a time, pH, and temperature-dependent manner. Active factor Xa generated on deacylation of anisoyl Xa exhibited amidolytic and prothrombinase complex activities in in vitro assays, the level being comparable to those of untreated factor Xa. When Anisoyl Xa was infused into rabbits, active factor Xa was generated on deacylation of the acylated enzyme, which shortened the activated partial thromboplastin time (APTT) in a dose-dependent manner. The duration of effect on rabbit APTT could be directly correlated to the level of human plasma factor Xa. Because anisoyl Xa bypasses the "tenase" complex that is compromised in hemophilia A and B and is unaffected by inhibitory antibodies, it has the potential to be used as an effective bypass therapy.

A comparative study of prothrombinase and thrombin inhibitors in a novel rabbit model of non-occlusive deep vein thrombosis.

A quantitative and non-occlusive deep vein thrombosis model was developed in rabbits. We used this model to test the antithrombotic activity of the prothrombinase complex inhibitors factor rXai and its chemical analog glutamyl-glycyl-arginyl chloromethyl ketone inactivated human factor Xa (EGR-Xai), along with the thrombin inhibitors D-phenylalanyl-prolyl-arginyl chloromethyl ketone (PPACK) and heparin. Dose dependent effects of the inhibitors during constant infusion were monitored. Measurements included thrombus weights, hemostatic parameters and both cuticle and ear bleeding times. In this model, factor rXai and EGR-Xai had comparable in-vivo efficacy, and showed 80%-93% inhibition at plasma levels of 6.5 nM (rXai) and 8 nM (EGR-Xai). Effects on ex-vivo clotting times varied among the inhibitors. At 80-100% thrombus inhibition, factor rXai and EGR-Xai had no statistically significant effect, while PPACK extended thrombin clotting time (TCT) times 2.3-fold, and heparin prolonged both activated partial thromboplastin time (APTT), prothrombin time (PT) and TCT ex-vivo clotting times 6.9-, 1.2-, and 7-fold respectively. At these dosages, cuticle and ear bleeding times were prolonged for all inhibitors and showed increases of 177%-389% (cuticle) and 45%-129% (ear). Our results demonstrate that direct inhibition of prothrombinase complex assembly is effective in arresting venous thrombosis.

Expression, purification, and characterization of inactive human coagulation factor Xa (Asn322Ala419).

We have expressed in Chinese hamster ovary cells a catalytically inactive form of human factor Xa (factor rXai). A recombinant precursor of human factor Xa was inactivated by two point mutations in the serine protease catalytic triad, Asp322Asn and Ser419Ala. A two-step purification to homogeneity of the secreted material involved immunoaffinity followed by heparin-agarose chromatography. Two forms were identified; a fully processed dimer (70%) and a partially processed monomer (30%). Limited N-terminal amino acid sequencing of factor rXai detected the predicted residues and gamma-carboxyglutamic acid content was 90% of human plasma control. Although devoid of measurable proteolytic activity, factor rXai competitively inhibited plasma factor Xa assembly into functional prothrombinase complexes (Ki = 3 x 10(-10) M). Factor rXai also inhibited plasma clotting in a dose-dependent manner. The possible use of recombinant catalytically inactive proteins as a general approach for pharmacological regulation of human diseases is discussed.

Pharmacokinetics of recombinant human tumor necrosis factor-alpha in rats. Effects of size and number of doses and nephrectomy.

The serum pharmacokinetics of recombinant human tumor necrosis factor-alpha (rHuTNF) were determined in male and female rats under various conditions. The clearance of rHuTNF after iv administration was determined to be saturable over the dose range of 10-63 micrograms/kg. Multiple iv administration did not significantly change the clearance of rHuTNF. Nephrectomy significantly reduced but did not eliminate the clearance of rHuTNF. The data suggest that sites other than the kidney also contribute to the elimination of rHuTNF. Female rats showed significantly reduced clearance of rHuTNF compared to male rats at all doses after single and multiple iv administration in control and nephrectomized animals.

Hypertonic sodium chloride solutions: effect on hemodynamics and survival after hemorrhage in swine.

We evaluated the hemodynamic response of 12% shed blood replacement with 7.5% sodium chloride after a nonlethal hemorrhage in chronically instrumented swine. Compared to no treatment or Ringer's lactate infusion, the hypertonic salt solution was followed by increased cardiac output. We then evaluated the effect of hypertonic sodium chloride on survival after an otherwise lethal hemorrhage in chronically instrumented and unanesthetized swine. Survival was significantly greater after replacement of 25% of the shed blood with 7.5%, but not with 0.9%, 5.0%, or 10.0% sodium chloride solution. The mechanism appears to be by increased tissue perfusion as indicated by lower lactate values. Resuscitation with low-volume hypertonic sodium chloride may be life saving and life supporting during the initial treatment of trauma patients when conventional therapy is not available.

Regional blood flow during hypothermic arrest.

Little is known about the efficacy of CPR in the setting of hypothermia-induced cardiac arrest. We measured organ blood flow produced by conventional closed-chest CPR in eight swine following normothermic KCl-induced cardiac arrest and in seven swine surface-cooled until cardiac arrest occurred. Radiomicrospheres were injected in the unanesthetized basal state, after five minutes of CPR, and after 20 minutes of CPR. After five minutes of CPR, the cardiac output and cerebral and myocardial blood flows (mean +/- SD) of hypothermic animals were 15.3 +/- 7.5 mL/min/kg, 0.16 +/- 0.11 mL/min/g, and 0.20 +/- 0.15 mL/min/g, respectively. Mean percentage flows were 7%, 15%, and 8%, respectively, of those measured in the unanesthetized prearrest state, and 50%, 55%, and 31%, respectively, of the flow produced during CPR in normothermic animals. Blood flow during hypothermic CPR did not change significantly over time; however, during normothermic CPR, cardiac output and cerebral and myocardial flows decreased so that at 20 minutes there were no significant differences from those values measured in hypothermic animals. The reduction in organ flow produced by external chest compression in hypothermic animals may be a result of the changes in the viscoelastic properties of the thorax that occur during profound hypothermia.

Fluid resuscitation after an otherwise fatal hemorrhage: II. Colloid solutions.

We developed a fixed-volume porcine hemorrhage model that simulates the rapid exsanguination of combat or civilian trauma victims. In this study we compared the ability of colloid resuscitation solutions to prevent death after an otherwise lethal hemorrhage in 100 swine. The shed blood was replaced in a 1:1 ratio with either autologous whole blood (WB), untyped swine fresh frozen plasma (FFP), typed FFP, 5% human serum albumin (ALB), or normal saline (NS). Survival rate analysis indicated that WB was significantly better than FFP (untyped), ALB, or NS but not better than typed FFP. The 24-hour survival rates were: WB = 90%, typed FFP = 79%, untyped FFP = 56%, ALB = 57%, and NS = 25%. All deaths in the untyped FFP group suddenly occurred during or within 15 minutes after treatment in a recovering animal. Deaths in the ALB group steadily occurred for up to 2 1/2 hours after treatment. Analysis of hemodynamic, arterial blood gas, and acid-base data indicated that WB and FFP provided a better acid-buffering capacity in surviving animals than NS or ALB. We conclude that compatible FFP is a better resuscitation agent than ALB after an otherwise fatal hemorrhage because FFP is a better acid buffer.